1. Field of the Invention
The invention relates to a process for preparing L-amino acids comprising a recombinant coryneform bacteria in which the AmtR regulator has been attenuated.
2. Discussion of the Background
L-Amino acids are used in human medicine, in the pharmaceutical industry, in the food industry and very particularly in livestock nutrition.
It is known that L-amino acids such as, for example, L-lysine are prepared by fermentation of strains of coryneform bacteria, especially Corynebacterium glutamicum. Because of their great importance, work to improve the methods used to prepare L-amino acids has been continuous. Process improvements may relate to fermentation technology measures such as, for example, stirring and supplying oxygen, or to the composition of the nutrient media, such as, for example, the sugar concentration during fermentation, or to the working up to product form by, for example ion exchange chromatography, or to the intrinsic output properties of the microorganism itself.
The methods used for improving the output properties of these microorganisms include mutagenesis, followed by selection and choice of mutants. The strains obtained in this way are resistant to antimetabolites or are auxotrophic for metabolites of regulatory importance, and produce L-amino acids. A known antimetabolite is the lysine analogue S-(2-aminoethyl)-L-cysteine (AEC).
Methods of recombinant DNA technology have likewise been used for strain improvement of L-amino acid-producing strains of the genus Corynebacterium, especially Corynebacterium glutamicum. These methods have been directed to modifying individual amino acid biosynthesis genes and investigating the effect on amino acid production.
Reference sources describing the biology, genetics and biotechnology of Corynebacterium glutamicum include in the “Handbook of Corynebacterium glutamicum” (Eds.: L. Eggeling and M. Bott, CRC Press, Taylor & Francis, 2005), the special edition of the Journal of Biotechnology (Chief Editor: A. Pühler) entitled “A New Era in Corynebacterium glutamicum Biotechnology” (Journal of Biotechnology 104/1-3, (2003)) and the book by T. Scheper (Managing Editor) “Microbial Production of L-Amino Acids” (Advances in Biochemical Engineering/Biotechnology 79, Springer Verlag, Berlin, Germany, 2003).
The nucleotide sequence of the genome of Corynebacterium glutamicum ATCC13032 is described by Ikeda and Nakagawa (Applied Microbiology and Biotechnology 62, 99-109 (2003)), in EP 1 108 790 and by Kalinowski et al. (Journal of Biotechnology 104(1-3), (2003)). The nucleotide sequence of the genome of Corynebacterium glutamicum R is described by Yukawa et al. (Microbiology 153(4):1042-1058 (2007)).
The nucleotide sequence of the genome of Corynebacterium efficiens is described by Nishio et al. (Genome Research. 13 (7), 1572-1579 (2003)).
The nucleotide sequences of the genome of Corynebacterium glutamicum and Corynebacterium efficiens are likewise available in the database of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, Md., USA), in the DNA Data Bank of Japan (DDBJ, Mishima, Japan) or in the nucleotide sequence database of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany or Cambridge, UK).
The structural definition of the Corynebacterium glutamicum genome made it possible inter alia to carry out wide-ranging investigations on the metabolism and regulatory network of this bacterium (Silberbach and Burkovski, Journal of Biotechnology 126(1): 101-110 (2006)).
An essential precondition for synthesizing amino acids and in general for growing the cells is an appropriate supply of nitrogen. C. glutamicum is able to utilize various nitrogen sources, including ammonium, L-glutamic acid, glutamine and urea. Depending on the concentration and nature of the available nitrogen source, particular enzymes and transport systems are synthesized and activated. For energy reasons, strict regulation is necessary (“nitrogen control”). The regulation of gene expression and the global signal transduction in the nitrogen metabolism of C. glutamicum has been investigated in detail by various authors.
The expression of nitrogen-regulated genes is regulated in C. glutamicum by the global repressor AmtR. When the nitrogen supply is good, AmtR represses expression of the genes of the amt-soxA-ocd operon, of the gltBD operon, of the amtB-glnK-glnD operon and of glnA and crnT genes (Jacoby et al., Molecular Microbiology 37: 964-977 (2000); Beckers et al., Microbiology, 147: 2961-2170 (2001); Nolden et al., FEMS Microbiological Letters 201: 91-98 (2001)). It was possible in further investigations (Beckers et al., Journal of Bacteriology 186(22): 7645-52 (2004); Beckers et al., Molecular Microbiology 58(2): 580-595 (2005)) inter alia to show an AmtR-dependent regulation also for the genes of the gluABCD operon, of the NCgl1915-1918 operon, of the urtABCDE operon, of the ureABCEFGD operon, and the codA gene and the NCgl1099 gene.
EP 1 460 128 reports on the effect of deleting the amtR gene in a ΔargR strain on the production of various amino acids.